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Pulmonary Pharmacology & Therapeutics xxx (2013) 1e4

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Pulmonary Pharmacology & Therapeutics

journal homepage: www.elsevier .com/locate/ypupt

Mucous hypersecretion and relationship to cough

Jay A. Nadel*

Cardiovascular Research Institute and Departments of Medicine, Physiology, and Radiology, University of California, San Francisco, USA

a r t i c l e i n f o

Article history:Received 11 December 2012Accepted 10 February 2013

Keywords:CoughMucinsMucous hypersecretionEGFRMucous pluggingPositive feedback

* University of California, 513 Parnassus Ave., Ro94143-0130, USA. Tel.: þ1 415 476 1105; fax: þ1 415

E-mail address: jay.nadel@ucsf.edu.

1094-5539/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.pupt.2013.02.003

Please cite this article in press as: Nadel JA, Mhttp://dx.doi.org/10.1016/j.pupt.2013.02.003

a b s t r a c t

A variety of foreign “invaders” such as viruses, bacteria and other particulates e.g., cigarette smoke, areinhaled, deposit on the airway surface and invade the “host.” Mucins produced by the surface airwayepithelium and by the submucosal glands are secreted into the airway lumen. Deposited particulatesadhere to the mucus and are cleared via mucociliary transport and via cough. Mucins are major con-stituents of mucus, which is important in the clearance of inhaled materials. Normally, secreted mucus iscleared without symptoms or interference with lung function. However, in obstructive airway diseasessuch as COPD, asthma, and cystic fibrosis, excessive mucus is produced. Because of the prominence ofmucous hypersecretion as a cause of cough, this discussion focuses on mechanisms regulating normalproduction of mucins and the mechanisms underlying exaggerated mucin secretion in chronicobstructive airway diseases. Mucins are produced by airway epithelial cells via a cascade of signals (theEpidermal Growth Factor Cascade) and secreted on the luminal epithelial surface, often in response tothe deposition of inhaled irritants. Normally, only minimal amounts of mucins are secreted, which assistin clearance of the inhaled particulates. However, in disease, additional pathways are induced via pos-itive feedback systems, which lead to mucous hypersecretion. In the large conducting airways, wherecough receptors are concentrated, mucous hypersecretion causes stimulation of neural receptors thatresult in cough. However, in small airways (e.g., bronchioles), because of their small diameters, mucoushypersecretion leads to plugging of the airways. Because there are so many small airways, their pluggingis difficult to detect early, and this locus is known as a “silent zone.” In chronic obstructive airway dis-eases, plugging of small airways may persist and increase over time, finally resulting in severe airwayobstruction. Different obstructive airway diseases induce inflammatory signaling (including mucoushypersecretion) via different stimuli, but often via similar signaling pathways. Application of presentknowledge of signaling that occurs with mucous hypersecretion can lead to novel therapies for hyper-secretion and cough induced in conducting airways and could prevent plugging in small airways that canlead to clinical deterioration and death.

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1. Roles of mucins in clearance of atmospheric “invaders”

Inhaled air contains a variety of fine particulates such as infec-tious agents, air pollutants, and allergens that are inhaled and de-posit on the airway epithelial surface. If the stimulus is irritatingand deposits on the surface of the large airways, the host respondsby secreting mucus and coughing, which assists in clearance of theirritant. The airway epithelium of the host produces mucins (amajor component of mucus), which adheres to the inhaled partic-ulates and assists in their clearance via mucociliary activity and viacough. In healthy individuals, clearance of invaders is usuallyaccomplished without symptoms or interference with lung

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ucous hypersecretion and re

function. However, in chronic obstructive airway diseases (COPD,asthma, cystic fibrosis), secretion may be exaggerated and causeserious disturbances in function that differ between the large andthe small airways.

2. Effects of mucin secretion in the large conducting airways

In healthy subjects, the airway epithelium of the conductingairways contains few mucins, and the airway lumen is usually freeof mucins, as shown in Fig. 1 (left). In individuals with fatal asthma,exemplified in Fig. 1 (right), goblet cell hyperplasia is seen and thelumen shows extensive staining for mucins (“mucous plugging”).

In the large conducting airways, mucus secretion and cough arelinked structurally. In Fig. 2, themajor (canine) airways are outlinedwith Tantalum (an inert, high contrast element) during anesthesia.The submucosal gland ducts and cough receptors both are located

lationship to cough, Pulmonary Pharmacology & Therapeutics (2013),

Fig. 1. Alcian Blue/PAS mucin staining in peripheral airway of control subject (left);arrows ¼ goblet cell staining and in patient with acute, fatal asthma (right);GC ¼ goblet cell. Reproduced by permission from Nadel, Burgel, Current Opinion inPharmacology, 1:254e258, 2001.

Fig. 3. Luminal contents in small airways stained with hematoxylin-eosin in a controlsubject with absence of luminal contents (left) and in a patient with Cystic Fibrosiswith lumens obstructed with “plugs” containing mucins (right). Adapted by permis-sion from Ref. [3].

J.A. Nadel / Pulmonary Pharmacology & Therapeutics xxx (2013) 1e42

in the large airways and both are concentrated at airway bi-furcations. Mechanical stimulation of the upper airways activatesthe afferent and efferent limbs of the vagus nerves innervating thesecretory cells, causing mucous hypersecretion. This effect wasblocked by cooling the vagus nerves or by administration of atro-pine. Thus, irritation of the upper airways causes reflex mucussecretion [1]. The co-localization of cough receptors and submu-cosal gland ducts facilitates cough clearance of mucus produced byglands. Thus, the particles of smoke that deposit on the surface ofconducting airways of chronic smokers can account for the coughand sputum production in chronic bronchitis. In addition, theconducting airways are normally the major sites of airway resis-tance. Here, mucous obstruction may contribute to dyspnea due totheir contribution to airway narrowing and to cough via theirstimulation of cough receptors. Stimulation of vagal sensory nervesalso causes reflex airway smooth muscle contraction, which couldalso play a role in airway narrowing [2].

3. Effects of mucin secretion in the small airways

In healthy normal control subjects, the production of mucins inthe small airways is sparse [3]. However, in chronic obstructiveairway diseases, obstructive changes in the small airways also occur.Here, Cystic Fibrosis (CF) is used as an example (Fig. 3). Lung tissueremoved at the time of transplantation shows extensive structuralchanges and plugging of small airways [3]. In the CF patient shownas an example, stained peripheral mucous plugs occupied a signif-icant percentage of the airway luminal volume (right), whereas acontrol subject showed almost no mucous staining (left). Thus,mucous plugs can play important roles in cystic fibrosis and in otherobstructive airway diseases, but these lesions can go unrecognized

Fig. 2. Co-localization of gland duct opening and cough receptors at airway bi-furcations. Tantalum bronchogram outlines airways. Arrow indicates site of submu-cosal gland duct opening and site of cough receptor concentration at large airwaybifurcation.

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until the majority of small airways are obstructed. Although thesurface epithelium of the small airways also produce mucins, theseairways differ from the large airways: (a) They do not contain coughreceptors, and therefore they are not likely to stimulate cough; (b)the small airways are very large in number, so total obstruction ofsome of these airways will not measurably increase airway resis-tance. Thus, because of paucity of symptoms, early detection ofobstruction of small airways does not occur.

Structural features of the small airways also contribute signifi-cantly to the difficulty in detecting their obstruction because: (a)They cannot be visualized by bronchoscopy; (b) they are generallybeyond the resolution of radiologic techniques; and (c) because theyare numerous and because they lack cough receptors, somepluggingof small airwayswill not contribute to detectable airway obstructionor cough. Nevertheless, chronic airway diseases including cysticfibrosis [3], fatal asthma [4], and COPD at the time of lung trans-plantation [5,6] show extensive plugging of small airways.

4. Strategies for treatment of mucous hypersecretion inairways depends on signaling pathways

Mucous hypersecretion in the conducting airways contributes todisturbing symptoms (chronic cough and sputum production). Evenmore seriously, mucous hypersecretion in the small airways leads tosmall airway plugging, which can result in progressive airwayobstruction, clinical deterioration and death. These events are wellestablished in cystic fibrosis [3] and in COPD [5] and have also beenshown to lead to death in acute asthma attacks [4]. This knowledgemakes it urgent to devise therapy for mucous hypersecretion.

5. Discovery of epidermal growth factor receptor regulationof mucin production

Serious research on mucins began at the end of the 20thcentury when a variety of stimuli were shown to induce mucin

lationship to cough, Pulmonary Pharmacology & Therapeutics (2013),

Fig. 4. Diagrammatic scheme of mucin production in human airways. The model is theresult of multiple studies that describe the “EGFR cascade.” A stimulus (e.g., a bacterialproduct) activates Dual Oxidase 1 (DUOX1) to form an active enzyme system forgenerating Reactive Oxygen Species (ROS, dark dots). ROS release activates the latentform of Tumor Necrosis Factor Alpha (TACE). TACE (represented by scissors) cleavesmembrane-bound Epidermal Growth Factor Receptor (EGFR) ligands, exemplified hereby pro-transforming growth factor-alpha (pro-TGF-a), releasing the soluble ligand(TGF-a), which binds to and activates EGFR by tyrosine phosphorylation. This results inactivating a series of cytoplasmic signals here represented by MAP Kinases (MAPK),with resultant signaling to the nucleus to produce and secrete mucins. This cascadealso produces a variety of other responses. Reproduced by permission from Ref. [17].

J.A. Nadel / Pulmonary Pharmacology & Therapeutics xxx (2013) 1e4 3

expression, such as ozone [7], acrolein [8], cytokines [9], neutro-phil elastase [10,11] and bacterial products such as Pseudomonas[12]. However, despite subsequent research, adequate treatmentof mucous hypersecretion still has not been accomplished. How-ever, over approximately the last decade, the discovery of asignaling cascade responsible for mucin synthesis has beendescribed and pathways responsible for exaggerated mucin pro-duction and secretion have been discovered. The discoveries ofthese cascades were derived from ideas originating from evolu-tion and animal migration from the sea: When animals migratedfrom sea to land, the need for enhanced oxygen uptake led to thedevelopment of the lungs as a mechanism for efficient oxygena-tion. Communication of the lungs with the ambient atmospherewas accomplished by the development of tubes (airways) thatconnected the lungs to the atmosphere. The ambient environmentis contaminated with a variety of particulates, (e.g., microorgan-isms, allergens, smoke). These foreign particulates enter the bodyduring inhalation and are deposited on the luminal surface of theairways, providing a means for invading the host. As a conse-quence, the cells on the surface of the airway epithelium havedeveloped a system for early defense against the invaders. Theseare referred to as “innate immune responses.” Thus, we realizedthat when an “invader” lands on the airway epithelial surface, theavailability of signaling messages for host defense on theepithelial surface are a key to winning the “battle” over the in-vaders. This led to a series of experiments that resulted inthe description of a surface signaling cascade that initiates asequence of events that provides host defense capable of defeat-ing the invader [13].

After examining the potential surface receptors, we hypoth-esized that activation of the epidermal growth factor receptor(EGFR), a major regulator of cell growth, could be responsiblefor mucin synthesis in the airway epithelium. Activation ofhuman airway epithelial cells with exogenous ligands (e.g., EGF)caused binding of the ligand to the receptor, which becameactivated and produced mucins. Selective inhibitors of EGFRactivation blocked the mucin response [14]. We showed thatcigarette smoke (CS)-induced mucin production was inhibitedby a selective inhibitor of EGFR activation [15]. Similarly, CS-induced mucin production was also abolished by a selectiveEGFR inhibitor in rats in vivo. Subsequent studies confirmed thatmucin production by multiple stimuli all depend on EGFRactivation [16].

6. Discovery of the epithelial surface cascade that regulatesmucin production

After the discovery that activation of EGFR on the airwayepithelial surface is responsible for mucin production [14], weinvestigated the surface signaling pathways that led from thedeposition of the inhaled stimulus on the airway epithelium to theactivation of EGFR, when the signaling enters the cell and signalsthe nucleus to produce mucin. When a foreign irritant deposits onthe airway epithelium, the host surface epithelial cells evoke aseries of signaling events known as the Epidermal Growth FactorReceptor Cascade that results in a series of defensive host re-sponses. Here, I describe briefly major elements of the cascade,using as the stimulus the deposition of a respiratory virus. Thesequence is outlined in Fig. 4.

6.1. Initial signal

Upon deposition, the virus is recognized by a pattern-recognition receptor. The airway epithelium expresses a toll-likereceptors (TLRs), which recognize specific pathogenic motifs.

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6.2. Generation of reactive oxygen species (ROS)

Dual Oxidase 1 (DUOX1) is expressed in airway epithelial cellsand generates ROS via NADPH oxidase activity [17]. In addition toROS generated by DUOX1, ROS can also be derived from otherstimuli such as cigarette smoke [18].

6.3. Tumor necrosis factor alpha converting enzyme (TACE) andother metalloproteinases cleave EGFR proligands

TACE is a member of the transmembrane metalloproteinasefamily. TACE is activated by ROS and cleaves EGFR proligands,allowing the free proligands to bind to and activate EGFR [19].

Activation of EGFR simulates a variety of downstream signals,resulting in a variety of products including mucin production [14].Following stimulation, EGFR signaling enters the cell. A variety ofresponses ensue depending on signaling partners. This variety ofdownstream partners and their responses are shown to be de-terminants of the exaggerated inflammatory responses inobstructive airway diseases.

In Fig. 4, a respiratory virus is shown as a stimulus for the pro-duction of mucins in the normal human airway epithelium. Notethat (a) the response to the stimulus proceeds with a series of cellsignals that results in limited mucin activation; (b) cell signalingproceeds via a stereotyped cascade that results from a variety ofstimuli; (c) the multi-signal cascade provides a variety of strategiesfor therapy. In normal individuals, the responses to a variety ofstimuli such as viruses, bacteria, allergens, cigarette smoke inairway epithelial cells is small and the invading stimulus is clearedfrom the body.

Because multiple stimuli (e.g., bacterial products) [12], cigarettesmoke [15], and neutrophil products [10,11] induce mucin pro-duction via an EGFR cascade, we hypothesized that exaggeratedmucin responses could be due to upregulation of selective signalingmolecules in individuals with mucous hypersecretion, while thesesignaling molecules remain quiescent in healthy individuals. Whena stimulus such as a respiratory virus infection activates EGFR toproduce mucin, a positive feedback occurs with EGFR rephos-phorylation, resulting in exaggerated mucin (and other responses).

Many GPCRs ligands induce EGFR activation. CCL20, a GPCRligand, is expressed in human airway epithelial cells [20], and itsexpression is increased in asthma [21], COPD [22], and cystic

lationship to cough, Pulmonary Pharmacology & Therapeutics (2013),

Fig. 5. Schematic of positive feedback loop between EGFR and CCL20/CCR6 signaling,leading to mucous hypersecretion. Activation of EGFR by an EGFR ligand (e.g., TGF-a)results in EGFR phosphorylation (py), activating intracellular signals exemplified bypERK 1, 2, which leads to mucin production. However, unlike normal cells, pERKactivation also leads to up-regulation and secretion of CCL20, a GPCR ligand that is up-regulated in chronic airway diseases. Secreted CCL20 binds to and activates its re-ceptor, CCR6. Activation of CCR6 leads to restimulation of an EGFR signaling cascade,causing further mucin production (MUC5AC), and resulting in mucous hypersecretion.EGFR ¼ Epidermal Growth Factor Receptor; MUC ¼ mucin; pY ¼ phosphorylation site;CCR6 ¼ A GPCR, ligand for receptor, CCR6. Reproduced by permission from Ref. [23].

J.A. Nadel / Pulmonary Pharmacology & Therapeutics xxx (2013) 1e44

fibrosis [20]. These findings make CCL20 binding to its receptor,CCR6, a potential mechanism for interactionwith the EGFR cascade.The sequence of the major elements in the EGFR signaling path-ways involved in mucous hypersecretion is described in Fig. 5.

When we stimulated EGFR directly with an EGFR ligand (here,TGF-a) in cells with exaggerated bronchial epithelial cell responses(NCI-H292 cells), EGFR activation occurred but then caused CCL20production, secretion and binding to its receptor CCR6. This led to afeedback loop with EGFR rephosphorylation and additional mucinproduction [23]. In normal human airway epithelial (NHBE) cells,TGF-a administration led to a single EGFR activation, with noupregulation of CCL20/CCR6 or EGFR reactivation. These findingsprovide a novel explanation for the mucous hypersecretion thatoccurs in obstructive airway diseases. Other molecules may also beinvolved in deleterious proinflammatory effects in the airwayepithelium and in other epithelial organs. For example, markedproinflammatory responses such as neutrophilic inflammation arefound in chronic obstructive airway diseases and stimulateneutrophilic inflammation via the production of interleukin 8 inairway epithelium [24,25].

7. Conclusions

Because of their importance in cough, herein is a brief dis-cussion of mucin regulation and its importance in airway dis-eases. Lungs were developed in land animals to allow oxygen tobe delivered to the host during inspiration. Particulate atmo-spheric contaminants are inhaled, deposit in the airways, andinvade the host. Cellular responses normally defend the host andkill and eliminate the invader. Defensive cellular responses areinitiated when the invader lands on the epithelial surface. Animportant signaling defensive cascade, known as the EpidermalGrowth Factor Receptor (EGFR) Cascade, generates airwayepithelial defensive responses including mucins, which normallyassist in the clearance of invaders. However, in variousobstructive airway diseases, exaggerated mucin responses cancause increased airway obstruction, especially during exacerba-tions, leading to clinical deterioration and death. Presently, thereis no adequate therapy for mucous hypersecretion. The EGFRcascade contains a variety of signaling molecules that couldblock these exaggerated responses. Because mucous hyperse-cretion occurs in a variety of obstructive airway diseases, and

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because their effects can be lethal, adequate therapy is sorelyneeded.

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lationship to cough, Pulmonary Pharmacology & Therapeutics (2013),